Fig. 6.
Mutation of positively charged residues predicted to interact with ECL2 impairs agonist affinity and alters receptor signalling in a pathway dependent manner. (A) Tops of TMs 2, 3, 4 and 5 of the apo GLP-1R TM bundle highlighting interactions between charged residues R3.30227, K4.64288, R5.40310 and residues located within ECL2 (R3.30227-D293, K4.64288-N304, R5.40310-N300). (B) TMs 2, 3, 4 and 5 of the GLP-1 docked activated GLP-1R TM bundle highlighting interactions between charged residues R3.30227, K4.64288, R5.40310 and residues located within ECL2 (R3.30227-D293, K4.64288- E292/N304, R5.40310-N300). Also shown is the GLP-1 peptide (dark red) with T11 that interacts directly with N300 located within ECL2. H7 of GLP-1 is also highlighted residing close to R5.40310. (C) Differences in equilibrium binding affinity (pKi) of mutant receptors relative to wildtype for GLP-1, oxyntomodulin and exendin-4. (D–F) Differences in the coupling efficiency (log τc) of GLP-1, exendin-4 and oxyntomodulin to three signalling pathways (cAMP production (D), pERK1/2 (E) and iCa2+ mobilisation (F)) at individual mutants compared to the wildtype receptor. These log τc were calculated from concentration–response curves presented in Fig. 2, Fig. 3, Fig. 4, and corrected for cell surface expression as measured by antibody labelling recorded in Table 1. Statistical significance of changes in affinity or coupling efficacy in comparison with wildtype were determined by one-way analysis of variance and Dunnett’s post test, and values are indicated with an asterisk (∗, p < 0.05). All values are ± S.E.M of four to six independent experiments, conducted in duplicate.